Proteomic Characterization of Isolated Retinal Pigment Epithelium Microvilli

Abstract

Polarized epithelial cells are characterized by displaying compartmentalized functions associated with differential distribution of transporters, structural proteins, and signaling molecules on their apical and basolateral surfaces. Their apical surfaces frequently elaborate microvilli, which vary in structure according to the specific type and function of each epithelium. The molecular basis of this heterogeneity is poorly understood. However, differences in function will undoubtedly be reflected in the specific molecular composition of the apical surface in each epithelial subtype. We have exploited a method for isolating microvilli from the mouse eye using wheat germ agglutinin (WGA)-agarose beads to begin to understand the specific molecular composition of apical microvilli of the retinal pigment epithelium (RPE) and expand our knowledge of the potential function of this interface. Initially, apical RPE plasma membranes bound to WGA beads were processed for morphological analysis using known apical and basolateral surface markers. The protein composition of the apical microvilli was then established using proteomic analysis. Over 200 proteins were identified, including a number of proteins previously known to be localized to RPE microvilli, as well as others not known to be present at this surface. Localization of novel proteins identified with proteomics was confirmed by immunohistochemistry in both mouse and rat eye tissue. The data generated provides new information on the protein composition of the RPE apical microvilli. The isolation technique used should be amenable for isolating microvilli in other epithelia as well, allowing new insights into additional functions of this important epithelial compartment. Molecular & Cellular Proteomics 3:1119 -1127, 2004.Epithelial cells are characterized by the asymmetric distribution of proteins and lipids in their plasma membrane: a basic feature referred to as polarity. The functional polarity of epithelial cells is dependent on the asymmetric distribution of specific enzymes, signaling molecules, and transport proteins between their apical and basolateral surface membranes (1). The apical surface of cuboidal and columnar epithelial cells commonly faces a luminal cavity and is characterized by the presence of numerous surface membrane elaborations referred to as microvilli. Microvilli greatly increase the apical surface area and, consequently, the number of transport and signaling proteins it contains, thereby enhancing the epithelial functional capacity. Absorptive epithelia such as kidney and intestine have their apical surface decorated with highly organized apical microvilli of uniform length and width. More dynamic and less organized structures are present in the epithelial cells of the placenta and the retinal pigment epithelium (RPE), 1 which perform endocytosis and phagocytosis, respectively. The basis of this morphological heterogeneity is poorly understood and is likely to be related to the specialized function and specific molecular composition of the ...

Annexin A2 Regulates Phagocytosis of Photoreceptor Outer Segments in the Mouse Retina

“…Because the apical processes are rich in actin, closure of the nascent phagocytic cup depends upon the reorganization of actin in conjunction with membrane fusion. Proteomic analyses of RPE and RPE apical processes have identified many actin modulators that may mediate these activities (West et al, 2003;Bonilha et al, 2004), including several members of the annexin family of Ca 2ϩ and phospholipid-binding proteins. Consistent with these reports, we observed upregulation of annexins A2 and A4 concomitant with acquisition of phagocytic competence in functionally differentiated ARPE19 cells (Turowski et al, 2004).…”

The Retinal Pigment Epithelium Apical Microvilli and Retinal Function

“…The RPE isolated microvilli are resolved by SDS-PAGE, in gel digested with trypsin, and peptides extracted and analyzed by mass spectrometry. 3,17 This procedure was done in mice eyecups with the RPE exposed and it has resulted in the identification of over 283 proteins, distributed over functional categories such as retinoid-metabolizing, cytoskeletal, enzymes, extracellular matrix components, membrane proteins and transporters, among others. A summary of selected proteins identified by this method is presented in Table 72.1 and has been recently described.…”

“…A summary of selected proteins identified by this method is presented in Table 72.1 and has been recently described. 3,17 The beads with the isolated RPE microvilli on their surface can be used for immunolabeling experiments, morphological (light and electron microscopy) as well as in biochemical experiments. In Figure 72.1 beads with isolated mouse RPE were fixed in 4% paraformaldehyde, permeabilized in triton X100, reacted with both a rabbit antibody to (A) protein kinase A regulatory subunit II (PKA RII ) and (B) a mouse antibody to protein kinase A regulatory subunit I (PKA RI ).…”

“…17 The cone and rod-associated matrix, present on top of the RPE apical surface, are firmly attached to the RPE apical surface. This is one of the reasons for the mass spectrometric identification of several novel extracellular matrix components such as fibromodulin, lumican, undulin 1, and neuroglycan C in the RPE isolated microvilli.…”

“…Carbonic anhydrase XIV was one of the proteins we identified by proteomic analysis in isolated RPE microvilli. 17 Aging studies have shown a decrease in both the number and the length of epithelial microvilli, and a declined function of plasma membrane enzymes and receptors. [26][27][28][29] Specifically, a decrease in the activity of some of the enzymes detected in RPE microvilli like Na,K-ATPase, LDH, glutathione S-transferase, phosphoglycerate kinase, adenylate kinase 30 and catalase has been established in various epithelia.…”